Isolation and characterization of polymorphic microsatellite loci in the Hawaiian flycatcher,the elepaio (Chasiempis sandwichensis)

Thirteen polymorphic microsatellite loci were isolated and characterized from two clades of an endemic Hawaiian flycatcher, the elepaio (Chasiempis sandwichensis). Seven dinucleotide repeats and one trinucleotide repeat were cloned from Kauai elepaio; five dinucleotide repeats were cloned from Oahu elepaio. Polymorphism was assessed in a sample of Oahu elepaio (n = 22) revealing two to 16 alleles per locus. Observed heterozygosity ranged from 0.14 to 0.91. However, linkage analysis exposed highly significant linkage disequilibrium between two of the most polymorphic loci. Twelve loci are therefore expected to be useful for investigations of population structure.     

Tetranucleotide microsatellite loci from eastern bluebirds Sialia sialis

We describe primers and polymerase chain reaction (PCR) conditions to amplify 18 tetranucleotide microsatellite DNA loci in eastern bluebirds (Sialia sialis). The primers were tested using individuals from two study sites in Georgia and South Carolina. Among individuals from the Georgia population (n = 23), the primer pairs developed in this study yielded an average of 6.6 alleles per locus (range 2–12), an average observed heterozygosity of 0.56 (range 0.24–0.96) and an average polymorphic information content of 0.65 (range 0.3–0.86). Among individuals from the South Carolina population (n = 19), the primer pairs yielded an average of 5.8 alleles per locus (range 2–9), an average observed heterozygosity of 0.56 (range 0.05–0.86) and an average polymorphic information content of 0.63 (range 0.29–0.83).     

Isolation and characterization of microsatellite markers from Pacific white shrimp (Litopenaeus vannamei)

We report the development of 11 polymorphic microsatellite loci in pacific white shrimp (Litopenaeus vannamei) using an unenriched genomic library. The number of the alleles ranged from two to 18 and observed hererozygosity ranged from 0.0286 to 0.9429, indicating that these markers will be useful for population studies and mapping in pacific white shrimp. Seven loci were detected deviated from Hardy–Weinberg, caused by deficiency of heterozygote, suggesting population genetic structure across the sampled population. No evidence for linkage disequilibrium was found.     

Characterization of polymorphic microsatellite loci for the polychaete tubeworm Hobsonia florida

Eight dinucleotide microsatellite loci were isolated and characterized from Hobsonia florida, a tube‐dwelling ampharetid polychaete. The identified loci were highly polymorphic, with allelic diversity ranging from six to 11 alleles. Levels of expected heterozygosity were 0.52 or greater in all cases, averaging 0.78 across the complete set of loci. Cross‐species amplification was successful in three of the eight loci for one or both of the other species (Melinna cristata and Ampharete acutifrons) tested. Although these novel loci were designed for immediate utility in H. florida population‐level research, these results indicate they may prove useful in studies of other related taxa.     

Development of species-specific PCR primer sets for the detection of Leptospira

Spirochetes of the genus Leptospira infect animals and humans and are the causative agents for the emerging infectious disease leptospirosis. Rapid and simple assays for the identification of individual Leptospira species are currently not available. For identification of individual Leptospira species, PCR primers that detect the ompL1 gene sequence for the majority of pathogenic leptospires were developed in this study. The primer pairs detect Leptospira interrogans, Leptospira borgpetersenii, Leptospira kirschneri, Leptospira santarosai, Leptospira weilii and Leptospira noguchii, without cross-reacting with other Leptospira species. The development of the primers revealed a divergence of the ompL1 gene within L. interrogans, splitting this species into two separate groups. The species-specific primers will be especially useful in epidemiological studies and disease outbreak investigations for the detection of Leptospira species in human, animal and environmental samples.     

A primer-based approach to genome walking

A genome walking strategy based on annealing and ligation of single-stranded DNA primers to 3′ overhangs following restriction endonuclease digestion was developed. A set of primers contains 4 nucleotides at the 3′ end that are complementary to overhangs formed by restriction endonucleasesApaI;PstI;SacI andSphI. Following ligation, 5′ end overhangs are formed on the DNA, which serves as sites for the adaptor primers and nested primers for PCR amplification in combination with the gene-specific primers. This strategy was verified by the amplification of up to 4 kb of a potato leafroll virus full-length infectious clone. The procedure could be adopted to target any upstream and downstream regions flanking known sequences within the plant genome.     

New Group-Specific 16S rDNA Primers for Monitoring Foaming Mycolata During Saline Waste-Water Treatment

Newly designed group-specific PCR primers for denaturing gradient gel electrophoresis (DGGE) were used to investigate foaming mycolata from a bioreactor treating an industrial saline waste-water. Genetic profiles on DGGE gels were different with NaCl at 1.65 and 8.24 g l⁻¹, demonstrating that mycolata community was affected by salinity. A semi-nested PCR strategy resulted in more bands in community genetic profiles than direct amplification. DNA sequencing of bands confirmed the efficacy of the novel primers with sequences recovered being most similar to foam producing mycolata. The new group-specific primers/DGGE approach is a new step toward a more complete understanding of functionally important groups of bacteria involved in biological treatment of waste-water. Revisions requested 1 December 2005; Revisions received 19 December 2005     

Development and evaluation of loop-mediated isothermal amplification assay for rapid and sensitive detection of canine parvovirus DNA directly in faecal specimens

Aims: To develop a specific and highly sensitive loop-mediated isothermal amplification (LAMP) technique for the rapid detection of canine parvovirus (CPV) DNA directly in suspected faecal samples of dogs by employing a simple method of template preparation. Methods and Results: LAMP reaction was developed by designing two sets of outer and inner primers, which target a total of six distinct regions on VP2 gene of CPV. The template DNA was prepared by a simple boiling and chilling method. Of the 140 faecal samples screened by the developed LAMP and the conventional PCR assays, 104 samples (74·28%) were found positive by LAMP, whereas 81 samples (57·85%) were found positive by PCR. The specificity of the LAMP assay was tested by cross-examination of common pathogens of dogs and further confirmed by sequencing. The detection limit of the LAMP was 0·0001 TCID(50) ml(-1) , whereas the detection limit of the PCR was 1000 TCID(50) ml(-1) . Conclusions: The developed LAMP assay detects CPV DNA in faecal specimens directly within an hour by following a simple and rapid boiling and chilling method of template preparation. The result also shows that the developed LAMP assay is specific and highly sensitive in detecting CPV. Significance and Impact of the Study: The result indicates the potential usefulness of LAMP which is a simple, rapid, specific, highly sensitive and cost-effective field-based method for direct detection of CPV from the suspected faecal samples of dogs.     

Significant interaction between LRP2 rs2544390 in intron 1 and alcohol drinking for serum uric acid levels among a Japanese population

A genome-wide association study identified that LRP2 rs2544390 in intron 1 was associated with serum uric acid (SUA) levels among Japanese, as well as polymorphisms of SLC22A12, ABCG2, and SLC2A9. This study aimed to confirm the association of rs2544390 C/T with SUA, as well as another LRP2 polymorphism (rs3755166 G/A) in the promoter. Subjects were 5016 health checkup examinees (3409 males and 1607 females) aged 35 to 69years with creatinine<2.0mg/dL. The subjects with SLC22A12 258WW, SLC2A9 rs11722228C allele, ABCG2 126QQ and 141Q allele (2546 males and 1199 females) were selected for analysis. Mean SUA was 6.03mg/dL for CC, 6.18mg/dL for CT, and 6.19mg/dL for TT among males (p=0.012), and 4.49mg/dL, 4.45mg/dL, and 4.42mg/dL among females (not significant), respectively. No association was observed for rs3755166. The association with rs2544390 was stronger among male drinkers. The odds ratio of drinking ≥5/week relative to no drinking for hyperuricemia (SUA≥7mg/dL and/or under medication for hyperuricemia) was 1.11 (95% confidence interval, 0.67-1.84) among CC males, 1.75 (1.22-2.51) among CT males, and 3.13 (1.80-5.43) among TT males. The interaction terms with drinking ≥5/week were 1.56 (p=0.156) for CT and 2.87 (p=0.005) for TT. This was the first report on the interaction between LRP2 genotype and alcohol drinking for SUA. Since the low density lipoprotein-related protein 2 (megalin) encoded by LRP2 is a multi-ligand endocytic receptor expressed in many tissues including the kidney proximal tubules, the association/interaction remained to be confirmed both epidemiologically and biologically.     

Unexpected problem in aphid DNA barcoding by universal primers

DNA barcode (mitochondrial COI) sequences have allowed for species identification of aphids. In this study, we newly found a DNA barcoding problem in a part of the DNA sequences for Sitobion avenae. Five S. avenae individuals showed differences of, on average, 32.60% in the DNA sequences from other conspecific individuals, and a BLAST search revealed that the five sequences are similar to those of aphid parasitoids such as Aphidius, Ephedrus and Praon spp. (Hymenoptera: Braconidae). Based on these results, we concluded that the universal primers used in aphid DNA barcodes can amplify barcode sequences from parasitoid species within host aphids.